The invention provides protein targets for disease intervention through inhibition of nucleic acid metabolism. Novel polypeptides for one such target, DNA-dependent ATPase A, and novel polynucleotides encoding DNA-dependent ATPase A are disclosed. The invention also provides compounds, including phosphoaminoglycosides, which act on such protein targets to inhibit nucleic acid metabolism. In addition, the invention provides screening assays for identifying compounds that inhibit nucleic acid-dependent ATPase activity, including, but not limited to, DNA-dependent ATPase A. Such compounds are useful in the treatment of diseases, including but not limited to cancer and infectious disease, through disruption of nucleic acid metabolism and induction of apoptosis. Moreover, the invention provides methods for prevention and treatment of diseases including, but not limited to cancer and infectious disease.
The interactions of proteins with nucleic acids involve a host of mechanisms for nucleic acid binding. Many nucleic acid-binding proteins (transcriptional repressors, transcriptional activators, restriction endonucleases, etc.) interact with a primary recognition sequence in a polynucleotide. These proteins: i) are generally classified as xe2x80x9csequence specific binding proteinsxe2x80x9d; ii) tend to bind double-stranded nucleic acids; and iii) tend to have significant numbers of contacts between their amino acid side chains and the edges of the bases which are exposed in either the minor or the major groove of a double-stranded nucleic acid. Proteins in this class have been the subject of extensive biochemical characterization and a significant number of protein-DNA co-crystal structures are now available (Steitz. Q. Rev. Biophys. 23, 205-280 (1990); Pabo and Sauer. Annu. Rev. Biochem. 61, 1053-1059 (1992)).
A second class of proteins, xe2x80x9cnonspecific binding proteinsxe2x80x9d (single-stranded DNA binding protein, DNA polymerases, etc.) are generally found to interact with single-stranded nucleic acids. The non-specific proteins are commonly considered to bind to a nucleic acid through predominately electrostatic interactions with the phosphodiester backbone of the nucleic acid and the favorable binding can be enhanced through protein-protein interactions (cooperativity). Biochemical analysis has been extensive for many of these proteins but unlike the sequence specific binding proteins, the information about protein-DNA contacts from crystallographic structures is very limited (Lohman and Ferrari. Annu. Rev. Biochem. 63, 527-570 (1994)).
Finally, there are a number of proteins that are not readily classified according to the specific or nonspecific categories. This third group of proteins is not generally grouped as a class but have the common feature of recognizing and binding to specific nucleic acid structures with neither the sequence specificity nor the electrostatic interactions of either group of proteins described above. This latter group would include proteins such as: i) E. coli RuvA and RuvB, which bind Holliday junctions and promote branch migration (Parsons et al., Proc. Natl. Acad. Sci. U. S. A. 89, 5452-5456 (1992); Muller et al., J. Biol. Chem. 268, 17185-17189 (1993)); ii) E. coli ribosomal protein L11, which recognizes the three-dimensional conformation of an RNA backbone and thus may regulate conformational changes during the ribosome elongation cycle (Ryan et al., J. Mol. Biol. 221, 1257-1268 (1991); Ryan and Draper. Biochemistry. 28, 9949-9956 (1989)); iii) topoisomerase II, which can yield cleavage of DNA following secondary structure-specific DNA recognition (Froelich-Ammon et al., J. Biol. Chem. 269, 7719-7725 (1994)); iv) DNA-dependent protein kinase, which phosphorylates proteins when activated by the presence of DNA double-stranded to single-stranded transitions (Morozov et al., Journal of Biological Chemistry. 269, 16684-16688 (1994); Chan and Lees-Miller. Journal of Biological Chemistry. 271, 8936-8941 (1996)); and v) transcription factor EBP-80, which also recognizes double- to single-stranded transitions in DNA (Falzon et al., Journal of Biological Chemistry. 268, 10546-10552 (1993)). The sequence specific binding proteins described above utilize a host of motifs for interacting with nucleic acids (zinc fingers, helix-turn-helix, xe2x80x9csaddlexe2x80x9d, etc.). Different potential motifs for this latter group of proteins have not yet been elucidated.
Nucleic acid-dependent ATPases are proteins that previously have not been generally classified as either specific or nonspecific binding proteins. Assays of helicases (molecular motors which unwind double-stranded nucleic acids) frequently require a structural element comprised of both a partial duplex nucleic acid and a nonhomologous tail on the strand to be displaced (Matson and Kaiser-Rogers. Annu. Rev. Biochem. 59, 289-329 (1990)). Furthermore, the hydrolysis of ATP by helicases leads to strand displacement (facilitated distortion) presumably through conformational changes in the helicase itself (Wong and Lohman. Science. 256, 350-355 (1992)).
Although nucleic acid-dependent ATPases have been identified, the precise role of these enzymes in nucleic acid metabolism has not been clearly elucidated. Moreover, nucleic acid-dependent ATPases have not been proposed as targets for therapeutic intervention through disruption of nucleic acid metabolism. Indeed, efforts into such intervention have focused on nucleotide analogs, such as ddI and AZT, which act on the polynucleotide chain itself in inhibiting DNA replication.
The present invention provides compositions and methods for preventing and treating disease through disrupting nucleic acid metabolism by targeting nucleic acid-dependent ATPase activity. The invention is based in part on the discovery, described below, of the role of a class of compounds known as phosphoaminoglycosides in inhibiting such nucleic acid-dependent ATPase activity. An understanding of the specificity of compounds that inhibit such activity, such as phosphoaminoglycosides, is derived from the underlying physico-biochemical principles of protein-nucleic acid interactions. Although the inventors are not required to provide an explanation of the underlying mechanism by which treatment and prevention are effected by the present invention, and without intending to be bound by any one particular mechanistic theory, the following discussion is provided regarding believed mechanisms of the invention. DNA-dependent ATPases are xe2x80x9cmolecular motorsxe2x80x9d that drive distinct cellular processes depending on the other protein domains or subunits with which they are associated. The concept of a molecular motor may be explained by a simple analogy. The molecular motor is analogous to the engine in a toy plane, boat or car. Each toy is composed of different parts brought together for different functions (flying, floating, rolling). The engine is common to each toy and provides the energy consumption which drives the function in each. Similarly, the DNA-dependent ATPase is the molecular motor equivalent to the engine. Multiple protein complexes are formed for each of the different DNA metabolic processes (e.g., DNA replication, DNA repair, transcription, recombination, chromatin remodeling, etc.) and the ATPase functions as a common core component (motor or engine) that drives the processes through the DNA-dependent consumption of ATP.
A further extension of this xe2x80x9cmolecular motorxe2x80x9d model is that disruption of the xe2x80x9cmolecular motorxe2x80x9d would lead to disruption of more complex processes. Disruption of nucleic acid-dependent ATPase activity, therefore, obtains the dual goal of cutting off the fundamental energy source for a number of nucleic acid metabolic processes, without general disruption of all ATPase functions within a living organism. Thus, in accordance with the invention, the energy supply for disease processes which involve relatively rapid nucleic acid metabolism (e.g., replication of infectious agent or cancer cell genetic material) is targeted; while the energy supply for other metabolic functions important to the treated subject (e.g., human, animal, or other vertebrate patient), left unaffected.
The invention is based, in part, on the discovery, described in detail below, of the protein:DNA interactions for DNA-dependent ATPase A. The structural and functional characteristics of DNA-dependent ATPase A activity described herein provides for the designing, testing, and use of therapeutic agents that specifically target these key energy-dependent nucleic acid metabolic processes. Such therapeutic agents that inhibit nucleic acid-dependent ATPases include, but are not limited to, phosphoaminoglycosides. Other researchers have failed to appreciate the importance of the phosphoaminoglycosides as chemotherapeutic agents.
The invention is further based, in part, on the discovery of the novel role of compounds in the inhibition of nucleic acid-dependent ATPase activity. Such inhibitory compounds, both known and novel, include but are not limited to phosphoaminoglycosides. The invention is also based, in part, on the discovery of genes, both human and bovine, encoding DNA-dependent ATPase A, and the recombinant production of a DNA-dependent ATPase polypeptide, as well as a detailed characterization of the activity and function of this polypeptide.
The present invention includes methods for disease intervention through inhibition of nucleic acid metabolism and induction of apoptosis. More specifically, the invention provides methods for prevention and treatment of diseases including, but not limited, to cancer and infectious disease including targeting the process of angiogenesis. The invention also provides compounds which act on such protein targets to inhibit nucleic acid metabolism. In addition, the invention provides screening assays for identifying compounds that inhibit nucleic acid-dependent ATPase activity. Such compounds are useful in the treatment of diseases, including but not limited to cancer and infectious disease. The invention provides protein targets for such intervention, which are used, in accordance with the invention, in screening assays to identify inhibitory compounds. The invention also provides polynucleotides encoding the protein targets of the invention, including novel DNA-dependent ATPase A polynucleotides.
The discovery of novel polynucleotides encoding both bovine and human DNA-dependent ATPase A is described in detail in the Example in Section 6, below.
The recombinant production and characterization of Active DNA-dependent ATPase A Domain (ADAAD) is described in detail in the Example in Section 7, below.
Methods for preparing phosphoaminoglycosides, and the ability of preparations of phosphoaminoglycosides to inhibit nucleic acid-dependent ATPases are described in detail in the Example in Section 8, below.
The Example in Sections 10 demonstrates the ability of phosphoaminoglycosides to disrupt DNA synthesis.
The Examples in Sections 11 and 12, below, demonstrate the ability of phosphoaminoglycosides to inhibit growth of prostate and breast cancer cell lines, respectively.
The Example in Section 13, below, demonstrates the ability of phosphoaminoglycosides to destroy tumors in mice in vivo.
The Examples in Sections 14 and 15, below, demonstrate the ability of phosphoaminoglycosides to inhibit growth and kill the protozoans amoeba and Leishmania, respectively.
The Example in Section 16 demonstrates that inhibition of DNA-dependent ATPase A activity disrupts DNA repair, respectively.